Beta-glucan in combination with anti-cancer agents affecting the tumor microenvironment

ABSTRACT

The present invention relates to the combination of soluble β-glucan and anti-cancer agents that affect the tumor microenvironment. Soluble β-glucan promotes an immunostimulatory environment, which allows enhanced effectiveness of anti-cancer agents.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/386,887, filed Dec. 21, 2016, which is a continuation ofInternational Application No. PCT/US2015/039977, filed Jul. 10, 2015;which claims priority to U.S. Provisional Patent Application Ser. Nos.62/022,754 filed Jul. 10, 2014; 62/076,094 filed Nov. 6, 2014;62/115,895 filed Feb. 13, 2015 and 62/149,892 filed Apr. 20, 2015, allof which are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

The present invention relates to combinations of soluble β-glucan andanti-cancer agents that affect the tumor microenvironment, includingimmunosuppression-relieving anti-cancer agents. β-glucan is a fungalPAMP and is recognized by pattern recognition molecule C3 in the serumas well as pattern recognition receptor, complement receptor 3 (CR3) onthe innate immune cells, including neutrophils and monocytes. β-glucan(β(1,6)-[poly-1,3)-D-glucopyranosyl]-poly-β(1,3)-D-glucopyranose), apolysaccharide β-glucan derived from yeast, is being developed as animmunotherapeutic agent in combination with anti-tumor monoclonalantibodies for the treatment of several cancers. β-glucan enables innateimmune effector cells to kill complement-coated tumor cells through acomplement CR3-dependent mechanism. Numerous animal tumor models havedemonstrated that administration of soluble β-glucan in combination witha complement-activating, tumor-targeting antibody results insignificantly reduced tumor growth and improved overall survivalcompared to either agent alone.

Cancers, however, are not just masses of malignant cells but complex“organs,” which recruit and use many other non-transformed cells.Interactions between malignant and non-transformed cells create thetumor microenvironment (TME). The non-malignant cells of the TME have adynamic and often tumor-promoting function at various stages ofcarcinogenesis. A complex and dynamic network of cytokines, chemokines,growth factors, and inflammatory and matrix-remodeling enzymes driveintercellular communication within the afflicted tissue. To effectivelybeat cancer, therefore, therapies must be developed to suppress thetumor-promoting nature of the TME.

SUMMARY OF THE INVENTION

This disclosure describes, in one aspect, uses and compositions ofsoluble β-glucan in combination with anti-cancer agents that affect thetumor microenvironment.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A-1D. Morphologic and functional characterization of in vitrocultured human M1 and M2 macrophages.

FIG. 2A-2D. Morphological, phenotypic and functional characterization ofsoluble β-glucan-treated M2 macrophages.

FIG. 3A-3D. Evaluations of CD4 T cell proliferation and modulation ofIFN-γ and IL-4 production in β-glucan-treated M1 and M2 macrophages fromhigh binders and low binders.

FIG. 4. Evaluations of T cell proliferation and modulation of IFN-γ inβ-glucan-treated M2 and M2a macrophages under immunosuppressiveconditions.

FIG. 5A-5E. Evaluations of β-glucan on CD4/CD8 T cell proliferation andactivation in the presence of Tregs.

FIG. 6A-6B. Characterization of in vitro cultured human immaturemonocyte-derived dendritic cells (imMoDC) and mature monocyte-deriveddendritic cells (mMoDC).

FIG. 7A-7D. Evaluations of β-glucan's effect on MoDCs maturation.

FIG. 8A-8C. Results of increased CD4 T cell proliferation by M2-β-glucandue to cell-to-cell contact.

FIG. 9. Results of increased CD4 T cell proliferation by M2-β-glucan dueto soluble factors.

FIG. 10. Analysis of β-glucan-treated M2 macrophages in high binders vs.low binders.

FIG. 11A-11B. Results of the functional evaluation of M2-β-glucanderived from low binders's monocytes in the presence of serum from ahigh binder.

FIG. 12A-12B. PD-L1 upregulation on β-glucan-treated M2 macrophagescultured in the presence of immunosuppressive cytokines (TCM).

FIG. 13. PD-L1 upregulation in MiaPaCa.

FIG. 14A-14B. Effects of soluble β-glucan on myeloid-derived suppressorcells (MDSC).

FIG. 15. Evaluation of β-glucan induced PD-L1 expression on tumor cells.

FIG. 16. Results of mouse study using IMPRIME PGG in combination withDC101 antibody.

FIG. 17A-17C. In vivo effect on tumor microenvironment of solubleβ-glucan and bevicizumab.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

β-glucans are polymers of glucose derived from a variety ofmicrobiological and plant sources including, for example, yeast,bacteria, algae, seaweed, mushroom, oats, and barley. Of these, yeastβ-glucans have been extensively evaluated for their immunomodulatoryproperties. Yeast β-glucans can be present as various forms such as, forexample, intact yeast, zymosan, purified whole glucan particles,solubilized zymosan polysaccharide, or highly-purified soluble β-glucansof different molecular weights. Structurally, yeast β-glucans arecomposed of glucose monomers organized as a β-(1,3)-linked glucopyranosebackbone with periodic β-(1,3) glucopyranose branches linked to thebackbone via β-(1,6) glycosidic linkages. The different forms of yeastβ-glucans can function differently from one another. The mechanismthrough which yeast β-glucans exert their immunomodulatory effects canbe influenced by the structural differences between different forms ofthe β-glucans such as, for example, its particulate or soluble nature,tertiary conformation, length of the main chain, length of the sidechain, and frequency of the side chains. The immune stimulatingfunctions of yeast β-glucans are also dependent upon the receptorsengaged in different cell types in different species, which again, canbe dependent on the structural properties of the β-glucans.

In general, β-glucan immunotherapies can include administering to asubject any suitable form of β-glucan or any combination of two or moreforms of β-glucan. Suitable β-glucans and the preparation of suitableβ-glucans from their natural sources are described in, for example, U.S.Patent Application Publication No. US2008/0103112 A1. In some cases, theβ-glucan may be derived from a yeast such as, for example, Saccharomycescerevisiae. In certain cases, the β-glucan may be or be derived fromβ(1,6)-[poly-(1,3)-D-glucopyranosyl]-poly-β(1,3)-D-glucopyranose, alsoreferred to herein as PGG (IMPRIME PGG, Biothera, Eagan, Minn.), ahighly purified and well characterized form of soluble yeast-derivedβ-glucan. Moreover, β-glucan-based immunotherapies can involve the useof, for example, a modified and/or derivatized β-glucan such as thosedescribed in International Patent Application No. PCT/US12/36795. Inother cases, β-glucan immunotherapy can involve administering, forexample, a particulate-soluble β-glucan or a particulate-solubleβ-glucan preparation, each of which is described in, for example, U.S.Pat. No. 7,981,447.

Anticancer immunotherapeutic drugs kill cancer cells through multiplemodalities: 1) direct activation of innate immune cells, 2) directactivation of adaptive immune cells, 3) indirect activation of bothinnate and adaptive immune cells by either making tumor cells moreimmunogenic or by subverting tumor-induced immunosuppression.

There is mounting evidence that myeloid cells at the tumormicroenvironment (TME), including M2 macrophages, N2 neutrophils andmyeloid-derived suppressor cells (MDSC), play a critical role in immunesuppression by directly causing functional exhaustion of the cytotoxic Tcells or by indirectly increasing the suppressive power of T-regulatorycells (Tregs). This leads to a skewed immunostimulatory versusimmunosuppressive balance in the TME. The immunostimulatory environmentof the TME is largely shaped by the presence of cytotoxic T cells and NKcells, cytolytic and phagocytosis-inducing M1 macrophages, cytotoxic N1neutrophils, humoral response inducing B cells, and antigen presentingimmunogenic dendritic cells (DC). Immunostimulatory cytokines andchemokines such as interferon gamma (IFN-γ), interleukin-12 (IL-12),tumor necrosis factor-alpha (TNF-α), etc. are key coordinators of theimmunostimulatory activity. Important players that bias theimmunosuppressive nature of the TME are anti-inflammatory Th2 cells, N2neutrophils, M2 macrophages, Tregs, and tolerogenic DC.Immunosuppressive cytokines and chemokines such as transforming growthfactor-beta (TGF-β), interleukin-10 (IL-10), macrophage colonystimulating factor (M-CSF), interleukin-4 (IL-4), etc. are keycoordinators of the immunosuppressive activity.

Soluble β-glucan, by virtue of being a pathogen associated molecularpattern (PAMP) that binds to CD11b on cells of myeloid origin, namelyneutrophils and monocytes, binds and increases the immunostimulatoryfunctions of N1 neutrophils and M1 macrophages and decreases theimmunosuppressive functions of MDSCs, N2 neutrophils and M2 macrophages.This modulation leads to cross-talk between the different innate andadaptive cell-subsets in the TME and eventually tilts the balancetowards immunostimulation. More specifically, once bound to peripheralblood monocytes, soluble β-glucan modulates the differentiation ofmonocytes to macrophages in M1/anti-tumorigenic versusM2/pro-tumorigenic polarizing conditions such that M1 polarization isenhanced which increases macrophage immunostimulatory functions and M2polarization is inhibited which decreases macrophage immunosuppressivefunctions. Soluble β-glucan directly affects M2 repolarization to the M1phenotype and drives Th1 polarization, and soluble β-glucan-primedinnate immune cells generate cytokines to indirectly affect CD4 and CD8T cell proliferation, even in the presence of Tregs, and eventuallydrive Th1 polarization.

Soluble β-glucan elicits an adaptive immune response via the two innatecell subsets that are known to bridge innate and adaptive immuneresponses, monocyte-derived macrophages and dendritic cells and willupregulate PD-L1 expression on both monocyte-derived macrophages anddendritic cells. In spite of PD-L1 upregulation, solubleβ-glucan-treated monocyte-derived macrophages and dendritic cellsenhance T cell activation and proliferation, and the coordinated immuneresponse elicited by soluble β-glucan elicits a tumor response akin toadaptive immune resistance, i.e., upregulation of surface expression ofPD-L1.

Soluble β-glucan can be combined with non-complement activating,tumor-targeting immune suppression-relieving MAbs. For example, solubleβ-glucan can be combined with anti-PD-L1 immune checkpoint inhibitors(Fc-engineered IgG1 MAb) in the treatment of several cancers, including,melanoma, renal cell carcinoma, lung cancer, etc. The efficacy ofanti-PD1/PD-L1 antibodies has been reported to be dependent upon theexpression level of PD-L1 on tumors. One of the mechanisms of PD-L1expression on tumors is called adaptive immune resistance where PD-L1expression is adaptively induced as a consequence of immune responseswithin the tumor microenvironment (e.g., interferon gamma production byactivated T-cells). Soluble β-glucan either directly, or indirectlyinduces Th1 polarization. This effect upregulates the expression ofPD-L1 on tumor cells, and thereby enhance the anti-tumor activity ofanti-PD1/PD-L1 antibodies. Examples of checkpoint inhibitors arenivolumab and pembrolizumab.

Soluble β-glucan can be combined with non-complement activating,non-tumor targeting MAbs that enhance immune co-stimulation. Forexample, a) anti-CD40 MAb (IgG2 MAb), targeting dendritic cells, b)anti-OX40, anti-41BB, enhancer of T-cells co-stimulation in thetreatment of several cancers.

Soluble β-glucan can also be combined with non-complement activating,non tumor-targeting immune suppression-relieving small molecules andnon-complement activating, tumor-targeting immune suppression-relievingsmall molecules. It can be used as an adjuvant in cancer vaccines todrive Th1 polarization. It can be used therapeutically to decreasesuppressive mechanisms in chronic diseases (i.e. TB) to hasten fullclearance of the infection. Lastly, it can be use to skew the Th2-Th1balance in Th2-dominant autoimmune diseases (allergies, asthma, atopicdiseases) to a Th1-polarized environment.

Although non-complement activating immune suppression-relieving agentsmay be preferred, especially for non-tumor targeting agents, theinvention may also be carried out with complement activating immunesuppression-relieving agents. One example may be bavituximab.

The invention includes, in part, co-administering a β-glucan withanother pharmaceutical agent, which, as used herein, may be an antibodypreparation or a small molecule preparation or any preparationadministered for affecting the TME. As used herein, “co-administered”refers to two or more components of a combination administered so thatthe therapeutic or prophylactic effects of the combination can begreater than the therapeutic or prophylactic effects of either componentadministered alone. Two components may be co-administered simultaneouslyor sequentially. Simultaneously co-administered components may beprovided in one or more pharmaceutical compositions. Sequentialco-administration of two or more components includes cases in which thecomponents are administered so that both components are simultaneouslybioavailable after both are administered. Regardless of whether thecomponents are co-administered simultaneously or sequentially, thecomponents may be co-administered at a single site or at differentsites.

In another aspect, the method includes administering to a subject acomposition that includes a β-glucan moiety conjugated to an antibody, atherapeutic antibody, an anti-tumor antibody or an antibody fragmentsuch as the Fc portion of an antibody. Modified and/or derivatizedsoluble β-glucan, including β-glucan conjugates of a β-glucan moiety andan antibody are described in International Patent Application No.PCT/US12/36795, which may also be applied to conjugates of antibodyfragments. The β-glucan moiety may be, or be derived from a β-1,3/1,6glucan. In this context, “derived from” acknowledges that a conjugatemay necessarily be prepared by creating a covalent linkage that replacesone or more atoms of the β-glucan. As used herein, “derived from aβ-1,3/1,6 glucan” refers to a portion of the β-glucan that remains aspart of a conjugate after replacing one or more atoms of the β-glucan toform the covalent linkage of the conjugate.

The β-glucan, the antibody or small molecule preparation, and/or thecombination of both components may be formulated in a composition alongwith a “carrier.” As used herein, “carrier” includes any solvent,dispersion medium, vehicle, coating, diluent, antibacterial, and/orantifungal agent, isotonic agent, absorption delaying agent, buffer,carrier solution, suspension, colloid, and the like. The use of suchmedia and/or agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the β-glucan or the antibody, its use in thetherapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the β-glucan and/or thepharmaceutical agent without causing any undesirable biological effectsor interacting in a deleterious manner with any of the other componentsof the pharmaceutical composition in which it is contained.

The β-glucan, the pharmaceutical agent, and/or the combination of bothcomponents may be formulated into a pharmaceutical composition. In someembodiments, the β-glucan and the pharmaceutical agent may be providedin a single formulation. In other embodiments, the β-glucan and thepharmaceutical agent may be provided in separate formulations. Apharmaceutical composition may be formulated in a variety of and/or aplurality forms adapted to one or more preferred routes ofadministration. Thus, a pharmaceutical composition can be administeredvia one or more known routes including, for example, oral, parenteral(e.g., intradermal, transcutaneous, subcutaneous, intramuscular,intravenous, intraperitoneal, etc.), or topical (e.g., intranasal,intrapulmonary, intramammary, intravaginal, intrauterine, intradermal,transcutaneous, rectally, etc.). A pharmaceutical composition, or aportion thereof, can be administered to a mucosal surface, such as byadministration to, for example, the nasal or respiratory mucosa (e.g.,by spray or aerosol). A pharmaceutical composition, or a portionthereof, also can be administered via a sustained or delayed release.

A formulation may be conveniently presented in unit dosage form and maybe prepared by methods well known in the art of pharmacy. Methods ofpreparing a composition with a pharmaceutically acceptable carrierinclude the step of bringing the β-glucan and/or the pharmaceuticalagent into association with a carrier that constitutes one or moreaccessory ingredients. In general, a formulation may be prepared byuniformly and/or intimately bringing the active compound intoassociation with a liquid carrier, a finely divided solid carrier, orboth, and then, if necessary, shaping the product into the desiredformulations.

The β-glucan, the pharmaceutical agent, and/or the combination of bothcomponents may be provided in any suitable form including but notlimited to a solution, a suspension, an emulsion, a spray, an aerosol,or any form of mixture. The composition may be delivered in formulationwith any pharmaceutically acceptable excipient, carrier, or vehicle. Forexample, the formulation may be delivered in a conventional topicaldosage form such as, for example, a cream, an ointment, an aerosolformulation, a non-aerosol spray, a gel, a lotion, and the like. Theformulation may further include one or more additives including such as,for example, an adjuvant, a skin penetration enhancer, a colorant, afragrance, a flavoring, a moisturizer, a thickener, and the like.

In some embodiments, the β-glucan may be derived from yeast such as, forexample, Saccharomyces cerevisiae. In some embodiments, the β-glucan caninclude a β-1,3/1,6 glucan such as, for example,β(1,6)-[poly-(1,3)-D-glucopyranosyl]-poly-β(1,3)-D-glucopyranose.

In some embodiments, the method can include administering sufficientβ-glucan to provide a dose of, for example, from about 100 ng/kg toabout 50 mg/kg to the subject, although in some embodiments the methodsmay be performed by administering the β-glucan in a dose outside thisrange. In some embodiments, the method includes administering sufficientβ-glucan to provide a dose of from about 10 μg/kg to about 5 mg/kg tothe subject, for example, a dose of about 4 mg/kg.

Alternatively, the dose may be calculated using actual body weightobtained just prior to the beginning of a treatment course. For thedosages calculated in this way, body surface area (m²) is calculatedprior to the beginning of the treatment course using the Dubois method:m²=(wt kg^(0.425)×height cm^(0.725))×0.007184. In some embodiments,therefore, the method can include administering sufficient β-glucan toprovide a dose of, for example, from about 0.01 mg/m² to about 10 mg/m².

In some embodiments, the method can include administering sufficientantibody that specifically binds the β-glucan to provide a dose of, forexample, from about 100 ng/kg to about 50 mg/kg to the subject, althoughin some embodiments the methods may be performed by administering theantibody in a dose outside this range. In some embodiments, the methodincludes administering sufficient antibody to provide a dose of fromabout 10 μg/kg to about 5 mg/kg to the subject, for example, a dose offrom about 100 μg/kg to about 1 mg/kg.

Alternatively, the dose may be calculated using actual body weightobtained just prior to the beginning of a treatment course. For thedosages calculated in this way, body surface area (m²) is calculatedprior to the beginning of the treatment course using the Dubois method:m²=(wt kg^(0.425)×height cm^(0.725))×0.007184. In some embodiments,therefore, the method can include administering sufficient antibody toprovide a dose of, for example, from about 0.01 mg/m² to about 10 mg/m².

In some embodiments, the β-glucan and pharmaceutical agent may beco-administered, for example, from a single dose to multiple doses perweek, although in some embodiments the method may be performed byco-administering the β-glucan and pharmaceutical agent at a frequencyoutside this range. In certain embodiments, the β-glucan andpharmaceutical agent may be administered from about once per year toonce per week.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements; the terms“comprises” and variations thereof do not have a limiting meaning wherethese terms appear in the description and claims; unless otherwisespecified, “a,” “an,” “the,” and “at least one” are used interchangeablyand mean one or more than one; and the recitations of numerical rangesby endpoints include all numbers subsumed within that range (e.g., 1 to5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

In the preceding description, particular embodiments may be described inisolation for clarity. Unless otherwise expressly specified that thefeatures of a particular embodiment are incompatible with the featuresof another embodiment, certain embodiments can include a combination ofcompatible features described herein in connection with one or moreembodiments.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1

Establishment and characterization of in vitro cultured human M1 and M2macrophages: CD14⁺ monocytes from human whole blood were enriched usingFicoll gradient and magnetic bead separation. Enriched monocytes (5×10⁵cells per mL) were then cultured in either M1-polarizing (XVivo 10 media(Lonza Group) supplemented with 5% autologous serum and 100 ng/mLrecombinant human granulocyte macrophage colony-stimulating factor(rhGM-CSF) (R&D Systems) or M2-polarizing (XVivo 10 media supplementedwith 10% autologous serum and 50 ng/mL recombinant human macrophagecolony-stimulating factor (rhM-CSF) (R&D Systems) conditions for 6 days.In experiments performed to evaluate the effect of β-glucan, whole bloodwas first incubated with vehicle (sodium citrate buffer) or 25 μg/mLsoluble β-glucan for 2 hours at 37° C. and then the monocytes wereisolated and differentiated. Morphology was checked before macrophageswere harvested for phenotypic analysis. Culture medium of the day 6macrophage culture (MCM) was collected, spun down to remove contaminatedcell pellet and then frozen down for subsequent cytokine analysis byELISA or used to setup a co-culture with CD3 & CD28-stimulated CD4 Tcells (MCM-CD4 T) for evaluation of either surface markers or CD4 T cellproliferation. The macrophages were used to setup a co-culture with CD3& CD28- or CD3 only-stimulated CD4 T cells (Mac-CD4 T) on day 6 forevaluation of either surface marker modulation or effect on CD4 T cellproliferation. For Mac-CD4 T cell proliferation study, M1 or M2macrophages were cultured with CD3 & CD28- or CD3 only-stimulated,CFSE-labeled, autologous CD4 T cells at a 1:10 ratio. T cellproliferation was measured at the end of the experiment (day 9-day 11)by flow cytometry, and results are graphically shown as CFSE-dilutionpeaks. The assessment of CD3-only stimulated T cells was always done onday 11. Quantitative results were reported as the Division Index (theaverage number of cell divisions a population underwent) calculated foreach of the triplicate wells in each of the culture conditions. Culturesupernatants of Mac-CD4 T co-cultures were collected for subsequentcytokine analysis.

For evaluation of Mac-CD4 T cell surface marker modulation, M2macrophages were co-cultured with T cells as described above and cellswere harvested on day 8, day 9 and day 10 to perform surface receptorstaining on both M2 macrophages and T cells.

For MCM-CD4 T cell proliferation study, CD3 & CD28-stimulated,CFSE-labeled CD4 T cells were cultured with 50% MCM. T cellproliferation was measured on day 11 as described above. Culturesupernatants of the MCM-CD4 T cell co-culture were collected forsubsequent cytokine analysis. MCM-CD4 T cell surface marker evaluationwas performed as described above.

M1 and M2 macrophages were prepared and characterized as described abovewere characterized for A) morphology, B) phenotype, C) functionalevaluation of Mac-CD4 T cell proliferation and D) cytokine analysis inthe co-cultures. FIG. 1A-FIG. 1C include representative results from 5different experiments.

As per literature, the morphology of M1 appeared more rounded and M2were more elongated fibroblast-like (FIG. 1A). Expression ofM1/M2-specific markers was evaluated by flow cytometry. Median MFI wascalculated for isotype control staining and surface antigen staining andthe results are shown in Table 1.

TABLE 1 CD274 HLA-DR CD163 CD14 CD206 CD209 CD80 CD86 (PD-L1) M1 Isotype91 91 23 151 127 151 127 91 control Surface 1715 148 100 859 1535 1792218 3570 antigen M2 Isotype 99 99 21 147 144 147 144 99 control Surface1054 1732 805 411 538 167 2463 385 antigen

Consistent with literature regarding phenotype, M1 macrophages typicallyexpressed higher levels of HLA-DR and CD274 (PD-L1), while M2macrophages expressed higher levels of CD163 and CD14. Additionally, incomparison to in vitro differentiated M2 macrophages, M1 macrophagesalso significantly helped CD4 T cells to proliferate as shown in FIG.1B. Concomitant with enhanced proliferation, increased production ofinterferon gamma (IFN-γ) was observed in the supernatants of M1 and CD4T cell co-cultures (FIG. 1C).

Steps for an alternative method for in vitro culture andcharacterization of human macrophages that includes activation of M1 andM2 macrophages (designated M1a and M2a macrophages) are outlined below.This methodology was used in the next series of experiments.

Activated M1a and M2s macrophages, prepared and characterized asdescribed above, were characterized for morphology and phenotype. Shownhere are representative results from 5 different experiments.

FIG. 1D shows the morphology of M1a and M2a macrophages. Expression ofM1a/M2a-specific markers was evaluated by flow cytometry. Median MFI wascalculated for isotype control staining and surface antigen staining andthe results are shown in Table 2.

TABLE 2 CD274 HLA-DR CD163 CD14 CD206 CD209 CD80 CD86 (PD-L1) M1aIsotype 123 123 23 119 79 119 79 23 control Surface 1535 812 191 10051215 537 3187 26135 antigen M2a Isotype 85 85 25 112 97 112 97 85control Surface 1800 3509 614 2186 4482 230 2445 4471 antigen

Example 2

Effect of β-glucan on M2 to M1 repolarization: M1 and M2 macrophagesfrom vehicle- or β-glucan-treated whole blood were prepared as describedabove. An expression of a panel of M1/M2-specific markers (includingHLA-DR, CD163, CD206, CD209, CD80, CD86 and PD-L1) were measured by flowcytometry. β-glucan pretreatment did not affect M1 macrophage phenotypebut did affect M2 macrophage phenotype. As shown in FIG. 2A, meanfluorescence intensity (MFI) of CD163 is downmodulated inβ-glucan-treated M2 macrophages. In addition, surface expression of CD86was enhanced as well as both protein and mRNA levels of PD-L1 (FIG. 2B).

Next, vehicle- or β-glucan-treated M1 or M2 macrophages were culturedwith CD3 and CD28-stimulated, carboxyfluorscein diacetate succinimidylester (CFSE)-labeled autologous CD4 T cells and T cell proliferation wasmeasured at the end of the experiment by flow cytometry and resultsquantitatively reported as division index (the average number of celldivisions a population has undergone). FIG. 2C is a representative CFSEdilution T-cell proliferation assay performed by co-culturing T-cellswith β-glucan-treated M2 macrophages, and the results show the abilityof the β-glucan-treated M2 macrophages to enhance CD4 T cellproliferation.

The culture supernatants from the CFSE dilution T-cell proliferationassay (FIG. 2B) were also measured for IFN-gamma levels by ELISA. FIG.2D is a representative graph of IFN-gamma levels showing a concomitantincrease in IFN-gamma production. Thus, β-glucan affects M2 to M1repolarization and drives anti-tumorigenic Th1 polarization.

Example 3

Effect of β-glucan on M2 to M1 repolarization in cells from high bindingsubjects vs. low binding subjects: Early studies evaluating binding ofsoluble β-glucan to neutrophils and monocytes revealed subjects havedifferent binding capabilities. Further studies found that solubleβ-glucan bound to at least some of high binding subjects immune cells,and high binding subjects also had higher levels of naturalanti-β-glucan antibodies. Functional studies identified general cut-offsof binding and antibody levels, which were used identify subjects ashigh binders (high response to β-glucan) and low binders (low responseto β-glucan).

To this end, evaluations of M1/M2 macrophages derived from solubleβ-glucan-treated monocytes from high binders and low binders werecarried out. M1 and M2 macrophages from high binders and low binderswere subsequently evaluated for A) phenotype, B) enhancement of CD4 Tcell proliferation and C) modulation of IFN-γ and IL-4 production. FIG.3A-FIG. 3C are representative results from 4 different experiments.

Expression of a panel of markers was evaluated by flow cytometry forvehicle- and β-glucan-treated, high binder-derived M1 and M2 macrophages(CD163 was evaluated twice). Median MFI was calculated for isotypecontrol staining and surface antigen staining and the results are shownin Table 3.

TABLE 3 High Binder CD163 CD163 CD274 HLA-DR (1) (2) CD206 CD209 CD86(PD-L1) M1 Isotype ctrl 58 58 103 84 98 139 58 Vehicle- 795 168 205 759404 2052 6130 β-glucan- 667 158 230 816 318 2113 5563 M2 Isotype ctrl 8686 91 122 104 254 86 Vehicle- 1259 2434 4079 1142 1064 2273 3087β-glucan- 1007 759 1153 1056 801 2953 4427

CD163 and CD86 were evaluated by flow cytometry for vehicle- andβ-glucan-treated, low binder-derived M1 and M2 macrophages. Median MFIwas calculated for isotype control staining and surface antigen stainingand the results are shown in Table 4.

TABLE 4 Low Binder CD163 CD86 M1 Isotype ctrl 58 278 Vehicle- 162 2860β-glucan- 132 3179 M2 Isotype ctrl 54 250 Vehicle- 3500 2445 β-glucan-3315 2284

The key result is that β-glucan-treated M2 macrophages had lowerexpression of CD163, one of the key M2 markers. Interestingly, thisresult was specific for high binders as expression of CD163 in remainedthe same between vehicle- and β-glucan-treated M2 macrophages.

Next, the ability of M1/M2 macrophages derived from solubleβ-glucan-treated monocytes from high binders and low binders to enhanceCD3 & CD28-stimulated CD4 T cell proliferation was evaluated. FIG. 3Ashows the results of the CD4 T cell proliferation assay in a high binderwhile FIG. 3B shows the results in a low binder. β-glucan-treated M2macrophages had significantly higher ability to enhance CD3 &CD28-stimulated CD4 T cell proliferation in comparison to that observedwith the vehicle-treated M2 macrophages in high binders while there wasno enhanced proliferation in low binders. In addition, β-glucan-treatedM1 macrophages showed no differences in this functional ability ascompared to the vehicle-treated M2 macrophages in either high binders orlow binders.

Modulation of IFN-γ and IL-4 production of vehicle- and β-glucan-treatedM2 macrophages was then evaluated. Concomitant with enhancedproliferation, significantly increased production IFN-γ, but not IL-4was observed in co-cultures of β-glucan-treated M2 macrophages and CD4 Tcells in high binders (FIG. 3C) but not in low binders (FIG. 3D). Thus,M2 macrophages derived from β-glucan-treated monocytes are M1-like inhigh binder subjects.

Example 4

Effect of β-glucan on M2 to M1 repolarization in immunosuppressiveconditions: Phenotypic and functional evaluation of β-glucan-treated M2aand β-glucan-treated M2 macrophages in the presence of immunosuppressivecytokines was carried out. M2 or M2a macrophages were prepared asdescribed above. On day 3, tumor-conditioned medium (TCM) was added M2macrophage cultures to account for 70% of the volume of the culture andthen evaluated for CD163 expression and functional activity on day 6.The TCM from BxPC3, a pancreatic cancer cell line, has been shown tocontain several immunosuppressive cytokines including M-CSF, TGF-beta,IL-4, etc. M2a macrophages were cultured in IL-4 as described above.

The β-glucan-treated M2a macrophages cultured in IL-4 and M2 macrophagescultured in TCM were first evaluated for CD163 and CD86 expression.CD163 and CD86 were evaluated by flow cytometry and median MFI wascalculated for isotype control staining and surface antigen staining andthe results are shown in Table 5.

TABLE 5 Immunosuppressive conditions CD163 CD86 M2a Isotype ctrl 149 147Vehicle- 664 4130 β-glucan- 309 4572 M2 Isotype ctrl 138 103 Vehicle-1736 1283 β-glucan- 1112 2697

As seen in previous M2 differentiation experiments using M-CSF, theβ-glucan-treated monocytes cultured in TCM also showed markeddown-regulation of CD163. In addition, the β-glucan-treated M2macrophages had higher HLA-DR expression (data not shown).

A functional evaluation of ability to modulate CD4 T cell proliferationwas then performed by CD4 T cell proliferation assay (three or sixreplicates in each condition). β-glucan-treated M2 macrophages culturedin TCM and M2a macrophages cultured in IL-4 maintained the ability toenhance CD4 T cell proliferation in comparison to that observed with thevehicle-treated M2 and M2a macrophages. Concomitant with enhancedproliferation, significantly increased production of IFN-γ was observedin co-cultures of macrophages and CD4 T cells (FIG. 4).

The above examples demonstrate that soluble β-glucan has the ability toinhibit M2 polarization and induce more M1-like cells as demonstrated bythe reduced expression of CD163, increased expression of CD86, and byinhibiting the ability of M2 to suppress CD4 T cell proliferation. Evenunder immunosuppressive conditions, simulated by the presence of eitherIL-4 in combination with M-CSF or tumor conditioned medium (TCM),soluble β-glucan was able to inhibit M2 polarization and enhance theirability to help CD4 T cell proliferation. The enhancement of CD4 T cellproliferation by M2-soluble β-glucan was accompanied with an increase inthe pro-inflammatory, Th1 polarizing cytokine, IFN-γ, and no change inthe production of immunosuppressive cytokine IL-4.

Example 5

Effect of β-glucan on CD4/CD8 T cell proliferation and activation in thepresence of Tregs: To obtain plasma, whole blood was treated for 6 hourswith 25 μg/mL β-glucan or vehicle, spun down and the plasma removed.50,000 autologous CFSE-labeled PBMCs were cultured in the treated plasmafor 3 days in the presence of 50,000 T cell activating CD3/28 beads(DYNABEADS Human T-Activator CD3/CD28 for T Cell Expansion andActivation). At the end of the culture, PBMCs were stained with CD4 andCD8 and T cell proliferation was measured by CFSE dilution. As shown bythe representative CFSE dilution plots in FIG. 5A, plasma fromβ-glucan-treated whole blood provided a significant enhancement in bothCD4 and CD8 proliferation as compared to vehicle-treated controls.

Next, to show the effect of soluble β-glucan on CD4 and CD8 cellactivation, the T cell proliferation assay described above was againcarried out. At day 3, however, the cells were stained for markers ofactivation including Granzyme B production and CD25 upregulation. Thegraphs shown in FIG. 5B demonstrate that β-glucan-treated plasmaenhances CD4 and CD8 cell activation.

The enhancement in proliferation was greatest when whole blood wastreated for the 6 hour incubation period prior to plasma isolation,indicating that this effect on T cell proliferation is the result of anindirect mechanism (i.e. cytokine release by innate immune cells). Todetermine whether the enhancement of T cell proliferation by β-glucan isdirect or indirect, T cell proliferation assays were carried out asdescribed above except the plasma from untreated (vehicle) whole bloodwas then either treated with β-glucan or vehicle prior to adding toautologous PBMCs. CFSE dilution was quantitated by Division Index usingFLOWJO software and plotted as fold change over vehicle control. Theresults shown in FIG. 5C indicate that β-glucan's enhanced effect is dueto indirect mechanisms.

Since these PBMC cultures contain Tregs, the suppressive ability ofTregs seem to be altered in the presence of soluble β-glucan and studieswere carried out to determine if β-glucan affected Treg suppression.Plasma from β-glucan-treated or vehicle-treated whole blood (describedabove) was added to 25,000 isolated CFSE-labeled autologous CD4 T cells(CD4⁺CD25⁻) along with increasing numbers of isolated autologous Tregs(CD4⁺CD25⁺) resulting in wells with increasing ratios. Cells were thenstimulated with 50,000 T cell activating CD3/28 beads for 3 days.Proliferation was subsequently measured by CFSE dilution and quantifiedby Division Index, which was used to calculate the % suppression ofTregs in the co-culture. % suppression=100−(Division Index of Tregwell/Division Index of 1:0 well)/100. The results are shown in FIG. 5D.Plasma from β-glucan-treated whole blood showed significant decreases inthe suppressive capacity of Tregs as compared to plasma fromvehicle-treated whole blood.

Treg suppression by β-glucan also resulted in enhanced IFN-gammaproduction. The Treg suppression assay was conducted as described above,and after 3 days of co-culture, supernatants were analyzed for IFN-gammaproduction. FIG. 5E shows the results of the IFN-gamma production fromwells cultured at an 8:1 T cell to Treg ratio. Taken together, theseresults show that β-glucan affects CD4 and CD8 proliferation along withTreg function resulting in enhanced anti-tumor adaptive effectorfunction.

Example 6

Establishment and characterization of in vitro cultured human immaturemonocyte-derived dendritic cells (imMoDC) and mature monocyte-deriveddendritic cells (mMoDC): Given that macrophages and dendritic cells arethe two key antigen presenting cell types that bridge innate andadaptive immunity, the phenotypic and functional effect of solubleβ-glucan was also evaluated on human monocyte-derived dendritic cells(MoDC). Monocytes enriched from soluble β-glucan- or vehicle-treatedwhole blood were cultured in media containing the appropriate cytokines,GM-CSF plus IL-4, for differentiation of dendritic cells. Steps includedin the method for in vitro culture and characterization of human MoDCsare outlined below.

imMoDCs and mMoDCs, prepared as described above, are shown in FIG. 6A.The morphology of mMoDCs is characterized by the presence of longprojections or dendrites.

mMoDCs were evaluated for CD80, CD83, CD86 and HLA-DR expression by flowcytometry, and median MFI was calculated for isotype control stainingand surface antigen staining and the results are shown in Table 6.

TABLE 6 CD80 CD86 CD83 HLA-DR MoDC Isotype ctrl 143 93 120 21 imMoDC 1651554 134 132 m □ □ □ □ 492 35637 448 470

mMoDC showed increased surface expression of the maturation andco-stimulatory markers CD80, CD83, CD86 as well as HLA-DR. Furthermore,these mMoDC also showed immunogenicity in an allogeneic mixed lymphocytereaction (four replicates in each condition), triggering increased CD4and CD8 T cell expansion (FIG. 6B).

Example 7

Effect of β-glucan on maturation of MoDCs: The phenotypic and functionalevaluation of mMoDC prepared from soluble β-glucan-treated whole bloodof a high binder and low binder was carried out. mMoDCs from a highbinder and a low binder were prepared as described above. mMoDCs wereevaluated for CD80, CD83, CD86 and HLA-DR expression by flow cytometry,and median MFI was calculated for isotype control staining and surfaceantigen staining and the results are shown in Table 7.

TABLE 7 -mMoDCs CD80 CD86 CD83 HLA-DR High Isotype ctrl 223 151 162 151binder Vehicle- 640 871 168 4286 β-glucan- 744 10759 466 7049 LowIsotype ctrl 213 141 162 30 binder Vehicle- 1056 67664 2346 3561β-glucan- 1197 77749 2924 2960

The increased expression of CD80, CD86, CD83 and HLA-DR onβ-glucan-treated mMoDC derived from high binders indicate that thesemMoDCs are more mature than those derived from low binders.

The β-glucan-treated mMoDC derived from high binders also showedincreased immunogenicity in an allo-MLR (four replicates in eachcondition), again triggering increased CD4 and CD8 T cell expansion(FIG. 7A) over cells derived from low binders (FIG. 7B).

In addition, the β-glucan-treated mMoDC derived from high binders wasable to modulate IFN-γ production over cells derived from low bindersand vehicle-treated mMoDCs (FIG. 7C).

MoDC derived from β-glucan-treated monocytes are more mature even inimmunosuppressive conditions. MoDCs were prepared as described above.TCM was added to account for 70% of the volume of the culture on day 0and was present throughout the culturing period. mMoDCs cultured in thepresence of TCM were subsequently evaluated for phenotypic changes.Median MFI was calculated for isotype control staining and surfaceantigen staining and results are shown in Table 8.

TABLE 8 Immunosuppresive Conditions CD80 CD86 CD83 HLA-DR MoDC Isotypectrl 120 88 118 31 Vehicle- 439 3340 274 352 β-glucan- 465 15797 607 361

Example 8

Cell-to-cell contact and soluble factors increase CD4 T cellproliferation by β-glucan-treated M2 macrophages: Using CD4 T cellproliferation as the read-out, the requirement of cell-to-cell contactor soluble factor(s) in initiating the proliferation by β-glucan-treatedM2 macrophages was studied. To study cell-to-cell contact betweenmacrophages and T cells, CD4 T cell proliferation was measured whenco-cultured with β-glucan-treated M2 macrophages in the absence of CD28co-stimulation and modulation of surface activation markers was studiedon both β-glucan-treated M2 macrophages and T cells in the co-culture.

Evaluation of cell-to-cell contact was carried out as follows: vehicle-and β-glucan-treated M2 macrophages and CD3 & CD28- versus CD3only-stimulated CD4 T cell co-cultures were utilized for measuring CD4 Tcell proliferation and IFN-γ production.

As shown in FIG. 8A, β-glucan-treated M2 macrophages cultured with CD4 Tcells in the absence of exogenous CD28 antibody demonstratedsignificantly higher ability to enhance CD4 T cell proliferation.Concomitant with enhanced proliferation, significantly increasedproduction of IFN-γ was observed in co-cultures of β-glucan-treated M2macrophages and CD4 T cells (FIG. 8B).

Changes in surface marker expression on both macrophages and CD3 &CD28-stimulated T cells were also measured. Vehicle- andβ-glucan-treated M2 macrophages and CD T cells from the co-cultures wereevaluated by flow cytometry for the modulation of co-stimulatory orco-inhibitory molecules. FIG. 8C and Table 9 are representative resultsfrom 2 different experiments.

TABLE 9 Surface Change in MFI on Change in MFI Markers Macrophages* on Tcells* HLA-DR No change NA CD86 Increase NA CD80 No change NA CD28 NA Nochange CTLA-4 NA No change CD40 No change NA CD40L NA No change 4-1BBLNo change NA 4-1BB NA No change OX40 No change NA PD1 (CD279) NAIncrease PD-L1 (CD274) Increase NA CD209 No change NA CD172 No change NA*Change in MFI on β-glucan-treated M2 macrophages/T cells relative tothat observed in vehicle-treated M2 macrophages/T cells

Of all the surface markers tested, a relative increase in surfaceexpression of CD86 (day 8) and PD-L1 (day 9) was observed on the surfaceof β-glucan-treated M2 macrophages as compared to that on thevehicle-treated M2 macrophages. Increased expression of PD-1 (day 9) wasobserved on the surface of T cells co-cultured with β-glucan-treated M2macrophages.

To determine whether soluble factors secreted from β-glucan-treated M2macrophages are required, measurements were carried out of CD4 T cellproliferation co-cultured with β-glucan-treated M2 macrophages MCM (50%of volume) and surface activation marker modulation on T cells incubatedwith the MCM were observed. FIG. 9 is representative of 2 differentexperiments. When evaluated by CD4 T cell proliferation assay,β-glucan-treated M2 macrophage MCM cultured with CD4 T cellsdemonstrated significantly higher ability to enhance CD4 T cellproliferation (FIG. 9) in comparison to the vehicle-treated M2macrophage MCM. In addition, the CD4 T cells cultured in vehicle- andβ-glucan-treated M2 macrophage MCM were evaluated for modulation ofco-stimulatory or co-inhibitory molecules (CD80, CD28, CTLA-4, 4-1BB andPD-1). Surprisingly, no change in any of the T cell markers was observed(data not shown).

Example 9

Analysis of β-glucan-treated M2 macrophages in high binders vs. lowbinders: As discussed previously, anti-β-glucan antibody (ABA)thresholds in subjects have been shown to be important for β-glucanimmunotherapy. Therefore, the importance of ABA threshold in β-glucan'sability to modulate M1/M2 polarization was investigated. β-glucan'sability to modulate M1/M2 polarization in high binders versus lowbinders was determined by both phenotypic and functional evaluations.

M2 macrophages from 4 high binders and 4 low binders were prepared andevaluated for their ability to modulate CD4 T cell proliferation. Thesupernatants from the various CD4 T cell proliferation conditions weremeasured for IFN-γ by ELISA. Results shown in FIG. 10 are representativefrom 4 different experiments. Fold change over the IFN-γ levels producedin the co-cultures of vehicle-treated M2 macrophages and CD4 T cells areplotted for each of the 4 donors.

In low binders, β-glucan did not modulate any of the phenotypic markerson the monocyte-derived macrophages in M1/M2 polarizing conditions (datanot shown), and in a functional evaluation of low binders by CD4 T cellproliferation assay, the β-glucan-treated M2 macrophages neitherenhanced CD4 T cell proliferation nor increased IFN-γ production.

Example 10

Serum cross-over studies: Because β-glucan failed to show modulation ofM1/M2 polarization in low binders, modulation by β-glucan usingmonocytes from a low binder in the presence of serum containing higherlevels of ABA (serum cross-over from a high binder) was evaluated. Totest this, M2 macrophages were prepared as described above with a fewmodifications. The whole blood of a low binder was spun down to removethe plasma and then the cells were reconstituted with serum obtainedfrom a high binder. The reconstituted blood was treated with vehicle orβ-glucan (25 μg/mL) for 2 hours at 37° C. The monocytes were evaluatedfor binding by using anti-β-glucan specific monoclonal antibody andsubsequent flow cytometry. The vehicle- or β-glucan-treated monocytes inwhole blood were then isolated and differentiated to M2 macrophages, andeither the M2 cells (data not shown) or the MCM were used to evaluatefor their ability to enhance CD4 T cell proliferation (six replicates ineach condition) and increase IFN-γ production using methods describedabove.

Monocytes in the whole blood of a low binder did not bind β-glucan butshowed significantly higher binding when a high binder's serumcontaining higher levels of ABA was added to the low binder's wholeblood (FIG. 11A). In addition, MCM from β-glucan-treated M2 macrophagesof a low binder crossed-over with a high binder's serum havesignificantly higher ability to enhance CD4 T cell proliferation incomparison to that observed with the vehicle-treated M2 macrophages.Concomitant with enhanced proliferation, significantly increasedproduction of IFN-γ was observed in co-cultures of β-glucan-treated M2macrophage and CD4 T cells (FIG. 11B).

Example 11

PD-L1 upregulation on β-glucan-treated M2 macrophages cultured in thepresence of immunosuppressive cytokines (TCM): Monocytes or M2macrophages were prepared as described above. On day 3, TCM was added toaccount for 70% of the volume of the culture and then evaluated forPD-L1 expression with TCM and then again when co-cultured with CD4 Tcells.

β-glucan-treated M2 macrophages cultured in the TCM had higher surfaceexpression of PD-L1 (FIG. 12A). There was also increased expression whenco-cultured with CD4 T cells (FIG. 12B).

Using the above system, it was determined that β-glucan has the abilityto inhibit M2 polarization as demonstrated by the reduced expression ofa key M2 marker, CD163, and by inhibiting the ability of M2 to suppressCD4 T cell proliferation. Even under an immunosuppressive environment,stimulated by the presence of either IL-4 in combination with M-CSF(data not shown) or tumor-conditioned medium (TCM), β-glucan was able toinhibit M2 polarization and enhance their ability to help CD4 T cellproliferation. The enhancement of CD4 T cell proliferation byM2-β-glucan was accompanied with an increase in the pro-inflammatory,Th1 polarizing cytokine, IFN-γ, and no change in the production ofimmunosuppressive cytokine IL-4. As expected with increased T cellactivation and IFN-γ production, increases in surface expression ofPD-L1 on β-glucan-treated M2 macrophages and PD-1 on T cells wereobserved. The β-glucan-treated M2 macrophages themselves, as well as thesoluble factors secreted by the cells are important for enhancing CD4 Tcell proliferation. Lastly, β-glucan inhibited M2 polarization in onlythe cells from healthy donors having higher levels of ABA.

Example 12

PD-L1 upregulation in MiaPaCa: β-glucan- and vehicle-treated M2macrophages and β-glucan-treated M2 macrophages+ABA were cultured withhigh binder serum and low binder serum to evaluate PD-L1 expression ontumor cells. FIG. 13 shows that β-glucan-treated M2 macrophagesincreased expression of PD-L1 on tumor cells in high binders, and withaddition of ABA, β-glucan-treated M2 macrophages also increasedexpression of PD-L1 on tumor cells in low binders.

Example 13

Effect of soluble β-glucan on myeloid-derived suppressor cells (MDSC):MDSC accumulate in the blood, lymph nodes, and bone marrow and at tumorsites in most patients and experimental animals with cancer and inhibitboth adaptive and innate immunity. MDSC are induced by tumor-secretedand host-secreted factors, many of which are pro-inflammatory molecules.The induction of MDSC by proinflammatory mediators led to the hypothesisthat inflammation promotes the accumulation of MDSC that down-regulateimmune surveillance and antitumor immunity, thereby facilitating tumorgrowth.

Blood was drawn at various times from a case study subject undergoingtreatment with IMPRIME PGG and analyzed for the presence of MDSC. Thefirst blood draw was done pre-infusion, cycle 8, day 1. As shown in FIG.14A, a large population of CD33⁺, MDSC are present in peripheral blood.A second blood draw was done post-infusion, cycle 8, day 1. As shown inthe second panel of FIG. 14A, within hours post-infusion the MDSCtransiently disappear. The last blood sample was drawn pre-infusion,cycle 8, day 15. The CD33⁺ MDSC are again present in the peripheralblood.

In another study, human cord blood was enriched for CD34⁺ cells andcultured for 9 days to produce CD33⁺CD11b⁺ cells (MDSC). The MDSC werethen treated with soluble β-glucan or citrate buffer (control) andevaluated for their ability to suppress T cell proliferation. The T cellproliferation assay was carried out at a 2:1 ratio of CD8 T cells totreated or untreated MDSCs. As shown in FIG. 14B, β-glucan-treated MDSCwere less suppressive to T cell proliferation.

These results indicate that β-glucan modulates the MDSC populationmaking them transiently leave the peripheral blood circulation and lesssuppressive to T cell proliferation. Thus, if one or more cancerimmunotherapeutic drugs or chemotherapeutic drugs are administered incombination with soluble β-glucan, especially during the period oftransiently loss of the CD33⁺ cell population, the therapies would bemore effective against the tumors.

Example 14

Supernatant from β-glucan-treated M2 macrophages/MoDC and T cellco-culture induces PD-L1 expression on tumor cells: M2 macrophages andMoDC were prepared as described above. The macrophages and the MoDC weresubsequently used in T cell proliferation assays as describedpreviously. The supernatants from these proliferation assays wereharvested and incubated with various tumor cell lines, including NSCLC,breast, pancreatic, colon, and B cell lymphoma. The expression of PD-L1on these tumor cell lines were evaluated post 48 hours by flowcytometry. Shown in FIG. 15 are representative results from 3 differentexperiments.

T cells require three signals for their effector mechanisms. Signal 1 isthe antigen presented in the context of MEW molecules on the antigenpresenting cells (APC), signal 2 is provided by the membranecostimulatory molecules on the APC, and signal 3 is the cytokinesproduced in the milieu for effector function. Coinhibitory molecules,such as PD-L1 can inhibit the effector functions of T cells.

Macrophages and dendritic cells derived from β-glucan-treated monocytesin vitro have higher expression levels of PD-L1, but the treatment alsoincreases the expression of the costimulatory molecule CD86, (signal 2),and cytokines (signal 3) allowing for enhanced T cell effector function.

The broader, innate and adaptive immune response elicited by β-glucanalso enhances PD-L1 expression on the tumor cell lines. These resultsdemonstrate that the up regulation of PD-L1 expression induced byβ-glucan on both the immune and the tumor cells makes it a promisingcombination partner with the checkpoint inhibitor cancer immunotherapy.

It is equally important to note that β-glucan also has the capability tooffset the inhibitory effect of PD-L1 up regulation by compensatorymechanisms such as increased expression of costimulatory molecules andproduction of immunostimulatory cytokines.

Example 15

Effect of soluble β-glucan in combination with anti-angiogenic agents onthe TME: Tumor angiogenesis alters immune function in the TME resultingin an immunosuppressive environment. Anti-angiogenic agents, such asanti-VEGFR2 antibody DC101 (mouse ramucirumab), have proven useful incancer therapy. Because soluble β-glucan can skew the TME to a moreanti-tumor environment, it was used in combination with DC101 to treatNCI-H441 non-small cell lung cancer (NSCLC) subcutaneous xenografts inmice to increase the effectiveness of the DC101 antibody.

6 to 8 week-old female athymic nude mice were injected with 5×10⁶ H441tumor cells in a volume of 0.2 ml subcutaneously in the flank. Mice weredosed biweekly when the mean tumor volume reached about 150 mm³ with thefollowing agents:

-   -   0.2 ml/mouse vehicle    -   1.2 mg/mouse IMPRIME PGG (Biothera, Inc.)    -   10 mg/kg or 20 mg/kg DC101 (Clone: DC101 Catalog #: BE0060)

Blood samples were collected on day 10 and 2 hours after the last dose.

Treatment groups included vehicle (PBS control), IMPRIME PGG alone,DC101 alone and DC101+IMPRIME PGG. Tumors were randomized to treatmentgroups once sizes reached a group mean of 150 mm³. The results of the 10mg/kg treatment groups are shown in FIG. 16.

As is evident from the graph, IMPRIME PGG+DC101 (acomplement-activating, non-tumor targeting antibody) actedsynergistically to minimize growth of the tumor. Thus, soluble β-glucanin combination with an anti-angiogenic agent (which may or may not be acomplement-activating, non-tumor targeting antibody) is an effectivecancer therapy.

Example 16

Soluble β-glucan in combination with anti-PD-L1 antibodies enhancestumor-free survival: In another animal study, mice were injected withMC38 tumor cells and randomized into treatment groups. 8 to 12 week-oldfemale C57BL/6 mice were injected with 1×10⁶ MC38 tumor cells, a colonadenocarcinoma that expresses low levels of PD-L1, in a volume of 0.1 mlinjected subcutaneously in the flank. Mice were dosed biweekly startingon day 3 with the following agents:

-   -   0.2 ml/mouse vehicle    -   1.2 mg/mouse IMPRIME PGG (Biothera, Inc.)    -   100 μg/mouse anti-PDL-1 Clone: 10F.9G2 BioXcell Catalog #:        BE0101

Blood samples were collected 1 hour prior to dose 1, 2 hours after dose3, the endpoint and 2 hours after the last dose (day 20). Treatmentgroups included vehicle (PBS control), IMPRIME PGG alone, anti-PD-L1antibody alone and anti-PD-L1+IMPRIME PGG. Tumors were randomized totreatment groups once sizes reached a group mean of 150 mm³. The resultsare shown in Table 10.

TABLE 10 Tumor-free Treatment Groups Survivors (day 29) Vehicle  1/18IMPRIME PGG  2/18 Anti-PD-L1  6/18 Anti-PD-L1 + IMPRIME PGG 14/17

Again, the combination of anti-PD-L1 antibody+soluble β-glucan workedsynergistically to effectively enhance tumor-free survival.

It should also be noted that PD-L1 expression on tumors is a biomarkerfor anti-PD-1 antibody responsiveness. Therefore, because solubleβ-glucan induces PD-L1 expression on tumors, soluble β-glucan will alsoenhance the effectiveness of anti-PD-1 antibodies. This is confirmed bythe increased PD-1 expression induced by soluble β-glucan treatmentdescribed above.

Example 17

In vivo effect on TME of soluble β-glucan and anti-angiogenic agents:Mice bearing H1299 NSCLC tumors were administered bevacizumab (ananti-angiogenic antibody) and IMPRIME PGG as described above for theother mouse studies. As shown in FIG. 17A, the treatment groupadministered the combination of bevacizumab and IMPRIME PGG showed anincrease in PD-L1 expression, FIG. 17B shows down-modulation of Arginase1 and FIG. 17C shows an increase in iNOS expression in the C11b positiveinnate immune infiltrate of the TME as compared to that of the groupadministered bevacizumab alone. Increased iNOS and decreased Arginase 1are markers indicating an M1, immunostimulatory environment. This dataclearly illustrates that soluble β-glucan increases the effectiveness ofanti-angiogenic agents and modulates the TME in vivo.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety. In theevent that any inconsistency exists between the disclosure of thepresent application and the disclosure(s) of any document incorporatedherein by reference, the disclosure of the present application shallgovern. The foregoing detailed description and examples have been givenfor clarity of understanding only. No unnecessary limitations are to beunderstood therefrom. The invention is not limited to the exact detailsshown and described, for variations obvious to one skilled in the artwill be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method of treating a subject having cancer, themethod comprising administering solubleβ(1,6)-[poly-(1,3)-D-glucopyranosyl]-poly-β(1,3)-D-glucopyranose and ananti-PD-L1 antibody.
 2. The method according to claim 1, wherein thecancer is melanoma, renal cell carcinoma, or lung cancer.
 3. The methodaccording to claim 1, wherein the cancer is breast cancer, pancreaticcancer, colon cancer, and B cell lymphoma.
 4. The method according toclaim 1, wherein theβ(1,6)-[poly-(1,3)-D-glucopyranosyl]-poly-β(1,3)-D-glucopyranose and theanti-PD-L1 antibody are in a single formulation.
 5. The method accordingto claim 1, wherein theβ(1,6)-[poly-(1,3)-D-glucopyranosyl]-poly-β(1,3)-D-glucopyranose and theanti-PD-L1 antibody are in separate formulations.
 6. The methodaccording to claim 1, wherein theβ(1,6)-[poly-(1,3)-D-glucopyranosyl]-poly-β(1,3)-D-glucopyranose isderived from yeast.
 7. The method according to claim 6, wherein theyeast is Saccaromyces cerevisiae.
 8. The method according to claim 1,wherein theβ(1,6)-[poly-(1,3)-D-glucopyranosyl]-poly-β(1,3)-D-glucopyranosestimulates the subject's immune system.
 9. The method according to claim1, wherein the anti-PD-L1 antibody is a non-complement-activatingantibody.
 10. The method according to claim 1, wherein the anti-PD-L1antibody is an Fc-engineered IgG₁ antibody.
 11. The method according toclaim 1, wherein the anti-PD-L1 antibody is an IgG₄ antibody.
 12. Themethod according to claim 1, wherein theβ(1,6)-[poly-(1,3)-D-glucopyranosyl]-poly-β(1,3)-D-glucopyranose and theanti-PD-L1 antibody are administered intravenously.
 13. The methodaccording to claim 1, wherein the method further comprisesadministration of a tumor targeting antibody.
 14. The method accordingto claim 1, wherein the method further comprises beta-glucan antibodies.15. The method according to claim 1, wherein the has high responsetoward solubleβ(1,6)-[poly-(1,3)-D-glucopyranosyl]-poly-β(1,3)-D-glucopyranose.
 16. Amethod of stimulating a subject's immune system against cancer cells,the method comprising administering solubleβ(1,6)-[poly-(1,3)-D-glucopyranosyl]-poly-β(1,3)-D-glucopyranose and ananti-PD-L1 antibody.
 17. The method according to claim 16, wherein theimmune stimulation comprises activation of M1 macrophages, N1neutrophils, NK cells, T cells, B cells or dendritic cells.
 18. Themethod according to claim 16, wherein the immune stimulation comprisesactivation of interleukin-12, interferon-γ, tumor-necrosis factor α, ora combination thereof.
 19. A method of removing immune suppression in atumor microenvironment, the method comprising administering solubleβ(1,6)-[poly-(1,3)-D-glucopyranosyl]-poly-β(1,3)-D-glucopyranose and ananti-PD-L1 antibody.
 20. The method according to claim 19, wherein themethod comprises suppressin of M2 macrophages, N2 neutrophils,myeloid-derived suppressor cells, or a combination thereof.